microorganisms Review

Bacterial Contaminants of Poultry Meat: Sources, Species, and Dynamics Amélie Rouger, Odile Tresse

ID

and Monique Zagorec *

Secalim, INRA, LUNAM Université, 44307 Nantes, France; [email protected] (A.R.); [email protected] (O.T.) * Correspondence: [email protected]; Tel.: +33-(0)2-40-68-7846 Received: 27 July 2017; Accepted: 21 August 2017; Published: 25 August 2017

Abstract: With the constant increase in poultry meat consumption worldwide and the large variety of poultry meat products and consumer demand, ensuring the microbial safety of poultry carcasses and cuts is essential. In the present review, we address the bacterial contamination of poultry meat from the slaughtering steps to the use-by-date of the products. The different contamination sources are identified. The contaminants occurring in poultry meat cuts and their behavior toward sanitizing treatments or various storage conditions are discussed. A list of the main pathogenic bacteria of concern for the consumer and those responsible for spoilage and waste of poultry meat is established. Keywords: chicken meat; bacteria; slaughter; spoilage; pathogen

1. Introduction Poultry meat consumption is steadily increasing worldwide; the last data available indicate it reached 14.2 kg per capita per year [1]. The developed western countries, particularly the United States of America (USA), are the largest consumers, with 49.8 kg per inhabitant per year [1]. The same trend of consumption increase is observed in the European Union (EU) and in countries of the Organization for Economic Co-operation and Development (OECD). Similarly, poultry meat consumption has doubled in France over the past 30 years and has become the second most consumed meat since 2012, reaching more than 26 kg per capita in 2014 (close to the consumption reported for the EU) after pork meat (32.5 kg per capita) [2]. Among poultry meat products, chicken carcasses, cuts, and processed products are the most consumed (~75% of total poultry meat) followed by turkey (~25%) and, to a lesser extent, duck [3]. In France, 60% of the chicken meat is sold as fresh cuts [2], often stored under various modified atmosphere packaging (MAP) [4]. Vacuum packaging, MAP, chilling, or marinades are different practices for ensuring microbial quality during the storage of poultry cuts, and depend on consumer habits and countries [4–7]. Therefore, ensuring the microbial safety of poultry meat products is an important issue in this context of increasing consumption and production. In fact, during and after slaughtering, the bacteria from animal microbiota, the slaughterhouse environment, and the equipment used contaminate carcasses, their subsequent cuts, and processed meat products. Some of these bacterial contaminants can grow or survive during food processing and storage. The resulting bacterial communities present in poultry meat can include pathogenic species such as Salmonella and Campylobacter, the two main pathogens responsible for human gastroenteritis due to poultry meat consumption [8]. Since 2005, Campylobacter has been the most commonly reported gastrointestinal bacterial pathogen in humans in the EU, where the numbers of reported confirmed cases in 2015 were 229,213 for human campylobacteriosis and 94,625 for human salmonellosis [8]. Poultry consumption has also been shown to be the first cause of foodborne outbreaks in the USA between 1998 and 2012 [9]. Other emerging pathogens, such as Aeromonas sp., may also be considered [10]. In addition to foodborne pathogens,

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bacteria responsible for spoilage may lead to large economic losses. Their growth and metabolic activity during shelf life leading to color, odor, taste, or texture defects are responsible for waste and losses of food products and have therefore an important impact on the economy of the poultry meat production sector. Most of the literature dealing with the microbial contamination of poultry meat is dedicated to detecting the presence of pathogens (mainly Salmonella and Campylobacter) and sometimes to studying their behavior under different decontamination, transformation, or storage conditions. Poultry meat contamination by spoilage bacteria has been less studied and is often limited to their enumeration by counting CFUs (colony forming units) on different, more or less specific, media. Challenge tests, based on the inoculation of individual strains or strain cocktails on meat cuts, have been used to investigate the growth ability of bacteria under various treatments. In contrast, only a few studies have recently used high throughput sequencing technologies to describe poultry meat contaminants, leading to their description at species level [6,7,11–13]. The aim of this review is to describe the state of the art regarding the knowledge available on the bacterial contaminants present in fresh poultry meat. The sources of contamination will be listed and the diversity of bacterial contaminants of poultry meat will be presented as well as their behavior along the production process, from the slaughterhouse to the end product, depending on the storage conditions or various treatments. 2. Sources of Contamination While muscles are sterile in healthy living birds, various microbiotas are hosted in the digestive tract, lungs, skin, feathers, etc. In slaughterhouses, the surfaces, air (aerosols), and liquids also encompass bacteria. Therefore, carcasses and cuts after animal killing can be contaminated by animal and slaughterhouse environment microbiota. Figure 1 summarizes the different steps in poultry slaughtering and the associated contamination routes. Although there are some differences between the practices in large-scale commercial slaughterhouses and small-scale slaughtering facilities, the main steps of poultry slaughtering are similar [14]. Compared to the slaughtering process of mammals, the main differences to be noted for poultry slaughtering are (i) the use of a water bath (hot or chilled) at different stages of the process; (ii) the feather removal step, which can be mechanical and is performed differently from removing the skin of mammals; (iii) the small size of birds (compared to cattle or sheep, for example) which has consequences on the ease of carcass manipulation and the mechanization of some processes. During the successive steps described in Figure 1, bacterial contamination may occur from equipment surfaces, water, and animal microbiota. Bacteria from the air and the environment can contaminate broiler meat [15]. The skin of poultry carcasses and cuts is directly in contact with air and equipment surfaces and is therefore easily contaminated. In fresh meat, bacteria are present on the surface rather than in the meat [16]. However, in processed products such as those which have been marinated, bacteria can migrate into the muscles [17]. Bacterial contamination by equipment surfaces can take place early in the process. For example, the rubber fingers used for feather removal or conveyor belts can be sources of bacterial contamination [18–20]. Even new rubber fingers can host bacteria and be a source of contamination for carcasses [19]. Cross contamination between carcasses or cuts may occur by direct contact or through contact with contaminated surfaces [20]. During the subsequent processing steps (deboning, cutting, mincing, and mixing) for meat-based foodstuff production, manipulators, air and equipment surfaces are the main sources of contamination. In fact, transformation operations increase the surface area of meat in contact with working surfaces and air [21]. Consequently, the level of bacteria is higher in transformed products than on primary cuts [21].

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Suspension Air Manipulators

Stunning

Killing

Scalding

Defeathering

Water

Rubber fingers

Chilling, calibration and packaging

Further transformation

Air, water, surfaces

Manipulators

Manipulators

Ingredients

Surfaces

Bleeding

Evisceration Animal microbiota Manipulators

Air, surfaces

Figure 1. Scheme representing the successive steps from poultry slaughtering to meat production and Figure 1. Scheme representing the successive steps from poultry slaughtering to meat production the associated contamination routes. After transport, the birds are suspended from the conveyor and and the associated contamination routes. After transport, the birds are suspended from the conveyor then stunned and killed. After bleeding, the birds are scalded in hot water at a temperature ranging and then stunned and killed. After bleeding, the birds are scalded in hot water at a temperature from 50 to 60 ◦ C to loosen the feathers. Subsequently, the feathers are mechanically abraded from ranging from 50 to 60 °C to loosen the feathers. Subsequently, the feathers are mechanically abraded the scalded birds. In large-scale slaughterhouses, feathers are removed using rotating rubber fingers from the scalded birds. In large-scale slaughterhouses, feathers are removed using rotating rubber and then the carcasses receive a spray wash prior to evisceration. Evisceration can be carried out by fingers and then the carcasses receive a spray wash prior to evisceration. Evisceration can be carried mechanical aspiration or manually after the carcasses have been cut open. At this stage, the gizzard, out by mechanical aspiration or manually after the carcasses have been cut open. At this stage, the heart, and liver are also retrieved. Next, the carcasses are chilled, either by immersion in cold water or gizzard, heart, and liver are also retrieved. Next, the carcasses are chilled, either by immersion in by air chilling. Subsequent transformation steps include cutting, deboning, grinding, and the use of cold water or by air chilling. Subsequent transformation steps include cutting, deboning, grinding, various treatments for meat product storage such as marinating or addition of different ingredients and the use of various treatments for meat product storage such as marinating or addition of (salt, spices) in processed products such as sausages. different ingredients (salt, spices) in processed products such as sausages.

The The water water baths baths used used during during the the process process have have aa washing washing effect effect that that diminishes diminishes the the bacterial bacterial loads, loads, but but can canalso alsopromote promotecross-contamination cross-contaminationbetween between carcasses carcasses[22,23]. [22,23]. The The high hightemperatures temperatures ◦ C) of the hot water used for scalding contribute to stopping bacterial growth. This helps to (50 (50to to60 60 °C) of the hot water used for scalding contribute to stopping bacterial growth. This helps to diminish onon skin. However, high temperatures dilate feather follicles and diminishthe thebacterial bacterialcounts countspresent present skin. However, high temperatures dilate feather follicles relax poultry skin. Further processing steps may therefore lead to bacteria transfer from feathers to skin and relax poultry skin. Further processing steps may therefore lead to bacteria transfer from feathers and follicles, previously dilated bydilated the hotby water, andwater, to entrapping bacteria after the cooling of plucked to skin and follicles, previously the hot and to entrapping bacteria after the cooling carcasses. Cold water used forwater chilling carcasses after evisceration as a cross-contamination of plucked carcasses. Cold used for chilling carcasses can afteractevisceration can act as a vehicle between carcasses, but also hascarcasses, a decontaminating effect by rinsing the surface ofrinsing carcasses, cross-contamination vehicle between but also has a decontaminating effect by the particularly when chlorine is added to the water as in the USA [24]. Although cold water and surface of carcasses, particularly when chlorine is added to the water as in the USA [24]. Although air chilling have different effects on diminishing Salmonella and Campylobacter counts, cold waterprocedures and air chilling procedures have different effects on diminishing Salmonella and no difference has been observed in the impact of the two procedures on the shelf life of cuts [24]. Campylobacter counts, no difference has been observed in the impact of the two procedures on the shelfThe life evisceration of cuts [24]. step, because of the microbiota present at high counts in the digestive tract, is a critical point of carcass step, contamination. tractatofhigh birdscounts hosts many The evisceration because ofThe the gastrointestinal microbiota present in the bacteria, digestiveincluding tract, is a some that can be potentially dangerous for the consumer such as Campylobacter spp. or Salmonella. critical point of carcass contamination. The gastrointestinal tract of birds hosts many bacteria, There is a some correlation between the number of Campylobacter in the such ceca as and the contamination including that can be potentially dangerous for the consumer Campylobacter spp. or level found on carcasses [25,26]. An average contamination level of 8.05 log CFU/g of ceca Salmonella. There is a correlation between the number of Campylobacter in the ceca andand the

contamination level found on carcasses [25,26]. An average contamination level of 8.05 log CFU/g of ceca and 2.39 log CFU/g of carcasses has been measured [25]. Poultry gut microbiota has been

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2.39 log CFU/g of carcasses has been measured [25]. Poultry gut microbiota has been studied in detail, particularly to correlate animal feeding, health, and gut microbiota [27–29]. However, to our knowledge, no study has yet been performed to establish a link between the composition of animal microbiota and that of the meat produced from these animals, although it has been reported that bacteria present in meat products originate at least partly from the animal digestive tract [13]. The evolution of the level of bacterial contamination throughout the slaughtering process has been described [22,30]. The contamination level of carcasses decreased after evisceration and chilling by immersion in cold water and increased again during storage at refrigerated temperature [30]. Similar results were observed by bacterial enumeration performed on neck skin [22]. This shows the washing effect at different steps, as well as the subsequent bacterial development that can occur during the storage period. After initial contamination, some bacteria can persist during meat product storage and can be recovered from killed animals before the scalding step, as well as after 10 days of the storage of carcasses at refrigerated temperature [30]. 3. The Major Bacterial Contaminants of Poultry Meat 3.1. Bacterial Contaminants The literature reports different practices for collecting bacteria from cuts or carcasses (rinsing, swabbing, stomaching) and the use of different media or incubation conditions which may lead to different results. Nevertheless, to illustrate the diversity of the bacteria targeted in the literature, we list the results (enumerations in log CFU/g) of several studies carried out by cultural methods on chicken meat (Table 1), resulting in a global inventory of the contaminants that can be encountered. Total viable counts represent various bacterial species, increasing during storage, and varying considerably between samples. As an example, we have previously shown that total viable counts from chicken legs sampled after storage at 4 ◦ C for 2/3rd of their shelf life varied from 3 to 8 log CFU/g [4]. Table 1. Values reported for various bacterial groups or species contaminating poultry meat at the beginning of the storage period. Values are expressed in log colony forming units (CFU)/g and have been collected from different reports. Reference 1 Total viable count Psychrotrophic bacteria Mesophilic bacteria LAB 3 Pseudomonas Enterobacteriaceae B. thermosphacta E. coli Coliforms S. aureus 1

[31] 5 ND ND ND 3.5 ND ND 2 2.2 ND

[32] 4.9 ND ND 3.9 4.2 ND 3 ND ND ND

[33] 2

ND 4.02−4.48 4.74−6.18 ND ND ND ND 0.70−2.34 3.54−4.64 0.68−2.43

[34]

[35]

[36]

[37]

4.88−5.41 ND ND ND ND 2.58−3.53 ND 2.60−3.63 ND ND

4.28 ND ND 3.66 3.38 ND 3.04 ND ND ND

5.66 ND ND ND ND ND ND ND 3.08 ND

ND 4.34 5.10 3.50 4.70 2.78 4.06 ND 2.86 ND

Data were collected from references [31–37]; 2 ND: not determined by the authors; 3 Lactic acid bacteria.

Pseudomonads, often recorded in poultry meat are mainly represented by the species Pseudomonas fragi, Pseudomonas lundensis, and Pseudomonas fluorescens [38,39]. Among Enterobacteriaceae, the main genera are Hafnia (Hafnia alvei, Hafnia paralvei), Serratia (Serratia fonticola, Serratia grimesii, Serratia liquefaciens, Serratia proteamaculans, and Serratia quinivorans), Rahnella, Yersinia, and Buttiauxella [40]. Several new Enterococcus or Lactobacillus species such as Enterococcus viikkiensis, Enterococcus saigonensis, and Lactobacillus oligofermentans have also been discovered in poultry meat products [41–43]. Brochothrix thermosphacta has also been often recorded in poultry meat. Among the various reports found in the literature, some targeted more specifically spoilage bacteria whereas others focused on pathogens.

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3.2. Spoilage Bacteria Growth of spoilage bacteria lead to defects in the products and can be responsible for unwanted taste, color, odor, texture, or aspect. There are multiple spoilage mechanisms, and they can result from the production of various metabolites such as volatiles or exopolysaccharides. Once bacteria contaminate meat and constitute the initial microbiota, the storage conditions and the various treatments applied shape the fate of this microbiota. The storage temperature as well as the nature and concentration of the gas used in gas mixtures for packaging are selective for some bacterial populations. Storage at low temperature favors the growth of psychrotrophic and psychrophilic bacteria while CO2 has an inhibitory effect on Pseudomonas spp. Some species can survive throughout the process, such as Shewanella putrefaciens, frequently found on carcasses during the slaughtering process and still present after 14 days of storage under air [30]. During storage, the bacterial load increases but the microbiota diversity decreases compared with that initially present [13,44]. Microbial spoilage occurs as a consequence of the growth and metabolic activities of spoiling bacteria. In most studies, the bacteria that dominate spoiled food have been considered those responsible for spoilage and, in some studies, the criterion of microbiological acceptability (total viable counts reaching 7 log CFU/g) has been used to define spoilage [44,45]. Examples of bacteria enumerations in chicken meat products considered as spoiled are listed in Table 2. Table 2. Enumeration of bacteria from spoiled chicken meat. Storage conditions and values of bacterial counts have been collected from different reports. Storage conditions

[45] 1

[36]

[35]

[31]

[32]

[46] 2

Duration (days) Temperature (◦ C) Packaging Total viable count 3 LAB Enterobacteriaceae B. thermosphacta Pseudomonas Coliforms

4 4 to 10 Air 7.55 8.04 8.36 ND 6 ND

5 7 Air 8.27 ND 4 ND ND ND 4.38

9 4 Air 7.55 7.02 ND 7.23 7.21 ND

11 3 70% CO2 , 15% O2 , 15% N2 6.5 ND ND ND 5 3.7

15 4 Air 8 7 6 7 6 ND

Until spoiled 6 65% N2 , 35% CO2 9.14 9.04 7.59 ND ND ND

1 Data were collected from references [31,32,35,36,45,46]; 2 Marinated poultry; 3 Bacterial counts are expressed as (log CFU/g), except in A (log CFU/cm2 ); 4 ND: not determined.

B. thermosphacta, lactic acid bacteria (LAB), Enterobacteriaceae and Pseudomonas spp. are considered potential spoilers of poultry meat. However, from the examples shown in Table 2, the involvement of these bacteria in spoilage could not be clearly concluded. Indeed, the variability in the microbial contaminants present in spoiled poultry meat illustrates the difficulty of clearly identifying the spoilage bacteria. Therefore, the definition of poultry meat spoilage bacteria must be considered carefully. A list of bacteria present in different meat products and their occurrence depending on the packaging atmosphere used for storage has been established by Doulgeraki and collaborators in 2012 [46]. Some of them were reported as poultry meat spoilage microorganisms. B. thermosphacta, P. fluorescens, and S. putrefaciens are among the spoilage bacterial species most cited in spoiled chicken meat products [23,30,45]. The spoilage potential of Aeromonas salmonicida, P. fluorescens, P. fragi and S. liquefaciens has also been evaluated by challenge tests and sensory evaluation [47]. Aeromonas hydrophila and Aeromonas sobria have been reported as psychrotrophic bacteria that could cause spoilage in addition to being potentially pathogenic for humans [30]. Molecular identification of colonies isolated from marinated spoiled poultry meat showed the involvement of several LAB species; in particular, Leuconostoc gelidum subsp. gasicomitatum and Lactobacillus oligofermentans [6,7,43,48]. Further investigation based on sensory analyses and genome or metabolic activity characterization of these LAB species confirmed their role in spoilage [49–51]. MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry) was also applied to colonies isolated

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from chicken breasts stored under two different MAPs and at two different temperatures in order to identify spoilage bacteria [44]. B. thermosphacta, H. alvei and bacteria belonging to the genera Carnobacterium, Janthinobacterium, Pseudomonas, and Serratia were identified among the dominant contaminants. However, in this study, spoilage was considered to occur when total viable counts reached 7 log CFU/g, with no indication about sensory deterioration [44]. 3.3. Pathogens Numerous articles have investigated the prevalence of various pathogens in poultry meat. Among these, Campylobacter and Salmonella make up a large majority of the reports. These two human pathogens can be present at high loads in the gastrointestinal tract of birds but, after contamination of poultry meat, it is important to detect their presence even at a very low level. Therefore, some studies have focused on establishing correlations between the occurrence in animals and in meat [25]. The emergence of antimicrobial resistance among foodborne pathogens is also extensively recorded [52]. In addition, the impact of breeding or farming on the prevalence and antibio-resistance in Campylobacter has been addressed [53]. Methods for the fast and accurate detection and identification of Campylobacter have been proposed [54]. Nevertheless, the data obtained by different methods should be carefully interpreted. As an example, the Campylobacter proportion enumerated in poultry feces determined either by high-throughput sequencing or by plating on various Campylobacter selective media gave quite different values [55]. Both Campylobacter jejuni and Campylobacter coli can be isolated from poultry meat [25], but also from human clinical cases that may result from contaminated food consumption [56]. No clear correlation could be established between the presence of Campylobacter in poultry meat and the level of bacterial contamination of chicken or turkey cuts [54]. Salmonella enterica is among the most tracked human pathogen, with the serovar Enteritidis being mainly associated with poultry meat and with outbreaks [57]. Other foodborne human pathogens present in various meat products have also been investigated, such as Listeria monocytogenes [58–62]. Listeria spp. prevalence in poultry meat is noticeable, with Listeria innocua as the dominant species followed by L. monocytogenes and several other Listeria species (Listeria welshimeri, Listeria grayi, and Listeria ivanovii). The prevalence of Staphylococcus aureus on poultry meat products has been addressed, although most of the literature has focused on antibiotic resistance and typing of the isolates [63–65]. Although there are a few reports on the detection of Clostridium perfringens on poultry meat [61], most of the literature focuses on assessing and modeling its growth on meat after spore germination following the slaughtering process [66–68]. Lastly, the emergence of Aeromonas from poultry meat products as a vector of human infection has also been reported [10]. Among Aeromonas spp. detected on poultry carcasses, Aeromonas caviae, A. hydrophila, Aeromonas salmonicida-masoucida, and Aeromonas schuberti have been reported to survive after 14 days of product storage [30]. 4. Variability of Bacterial Communities Regarding Different Matrices and Processes We have combined the data reported in the literature to draw a picture of the bacterial contaminants occurring in poultry meat depending on different variables. We chose to select variations depending on the meat matrix or on the storage/transformation process. The bacterial contaminants present in poultry carcasses and cuts and their growth depend on different factors: the storage temperature, the gas composition used for MAP, the composition of marinades or various chemical treatments that can be applied to control bacteria. A number of studies were selected to illustrate the diversity of the methods used. 4.1. Variability of Bacterial Contaminants Regarding Meat Matrix and Origin Most of the literature focuses on chicken meat and, to a lesser extent, turkey meat. A comparative study of the microbiological quality of poultry meat in Morocco showed that turkey meat was more contaminated (5.4−7.4 log CFU/g total aerobic counts) than chicken meat (4.5−6.6 log CFU/g) [61]. Nevertheless, for several pathogens (Escherichia coli, S. aureus, and C. perfringens) the contamination

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level was similar in chicken and turkey meat. The difference might result from the different farming conditions and/or intrinsic differences between these two birds. These authors also noticed that the traditional slaughtering process increases contamination by microbial communities. This correlates with another observation, which reported a higher contamination level of skins of chicken carcasses from traditional markets and artisanal slaughterhouses [33]. This study, also carried out in Morocco, showed higher counts of mesophilic and psychrotrophic bacteria, total and fecal coliforms, and S. aureus on artisanal products than on carcasses purchased from supermarkets. The contamination level regarding different cuts or raw vs. transformed products has also been evaluated. The mesophilic bacteria from various poultry cuts (thighs, wings, giblets, hamburgers, and sausages) were enumerated These were higher in processed products (hamburgers, sausages) with approximately 7 log CFU/g, than in the fresh cuts (thighs, wings) with approximately 5.7 log CFU/g [21]. This may result from the temperature during the transformation process (10 ◦ C) and from the mixing steps that increase the surface area of meat in contact with surfaces and air, both favorable to bacterial growth and to the possibility of increased contamination. 4.2. Variability of Bacterial Contaminants Regarding Storage Temperature The importance of temperature for bacterial growth can be assessed at different critical points between the slaughtering and the consumption of the product, in particular (i) during carcass handling (the temperature in the processing plants is usually about 10 ◦ C); and (ii) during the storage of meat products (with an estimation of a rupture in the cold chain between the time of sale and the consumer’s fridge, whose temperature is estimated to be higher than 4 ◦ C). The effect of chilling carcasses using chilled air or a cold water-bath on their microbial contaminants has been assessed [69]. Refrigeration by chilled air slows down the development of the total viable count (approximately 1 log) and causes a rapid decrease in temperature. This inhibits the multiplication of Salmonella and Campylobacter, thus chilled-air cooling would be more efficient. However, it is necessary to take the fact that Listeria can grow at this storage temperature into account. The product shelf life can be increased by storage at low temperature and the absence of a break in the cold chain [70]. The shelf life can even be doubled when the temperature is lowered to 3.4 ◦ C compared to storage at 8.3 ◦ C. Low temperatures delay the growth of Enterobacteriaceae, which can produce sulfuric compounds and the organoleptic deterioration of the meat quality. On the other hand, the growth of psychrotrophic bacteria is enhanced; at 4 and 7 ◦ C, the total viable counts develop faster than at 0 ◦ C [69]. A faster development of total viable counts is also observed at 10 ◦ C when compared to 4 ◦ C [45]. Storage at 4 ◦ C is damaging for B. thermosphacta and S. putrefaciens growth after 7, 10 or 14 days whereas A. hydrophila and A. sobria are psychrotrophic bacteria that can develop at low temperature [30]. Smolander et al. (2004) also pointed out, however, that the shelf life of products cannot be lengthened too much by storage at 0 ◦ C, because pathogenic agents such as Listeria can multiply at these temperatures [70]. 4.3. Variability of Bacterial Contaminants Regarding the Packaging Gas Composition Dioxygen-enriched atmospheres have been largely used for ensuring typical red meat color during storage. However, poultry cuts may be considered differently [71] and different gas compositions are in use for poultry cuts [4,71]. CO2 has a bacteriostatic effect, which inhibits the growth of aerobic microorganisms such as Pseudomonas spp. that are considered putative spoilage organisms. The effect of different gas compositions on microbial contaminants has been evaluated [35,72]. As shown in Table 3, the storage period to reach spoilage (estimated as the time for total viable counts to exceed 7 log CFU/g) can be prolonged by CO2 -enriched atmospheres when compared to storage under air. The shelf life before spoilage occurrence can be extended from six days under air to 12 and 15 days under MAP with 30% CO2 –70% N2 and 70% CO2 –30% N2 , respectively [35] or from 5 to 8 days with 30% CO2 −70% N2 [72]. B. thermosphacta, Pseudomonas, and Enterobacteriaceae counts did not seem significantly affected by the type of packaging but were detected as bacteria responsible for spoilage [32]. LAB counts

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varied depending on gas composition, but also between the studies [35,72]. Prolonged shelf life by CO2 enriched atmospheres (30% CO2 –70% N2 , 60% CO2 –40% N2 , and 90% CO2 –10% N2 ) has also been reported for precooked chicken breasts [73]. Table 3. Comparison of bacterial counts at spoilage time after storage under air or under two different MAPs. Days to Reach Spoilage (Total Viable Count ≥ 7 log CFU/g) Air

Air

61

52

30% CO2 −70% N2 12 1

30% CO2 −70% N2 82

70% CO2 30% N2 15 1

Bacterial Counts (log CFU/g) When Spoilage Was Reached LAB Pseudomonas B. thermosphacta Enterobacteriaceae 1

2.91 6.28 6.90 6.15

3.2 6.2 ND 3 5.8

6.88 6.83 6.39 6.42

4.1 6.1 ND 5.8

6.89 6.71 7.21 6.71

Data were collected from [35]; 2 Data were collected from [72]; 3 ND: not determined.

Replacing nitrogen with argon in the composition of MAP (proportion from 15% to 82%) was tested [74]. No strong difference was observed, with only B. thermosphacta appearing to be significantly affected by a high proportion of Ar in the gas mixture. Nevertheless, the various proportions of Ar or N2 /CO2 /O2 in the gas mixtures tested shaped differently the growth dynamics and the ratio of different populations (LAB, B. thermosphacta, Pseudomonas spp., and Enterobacteriaceae). The growth of mesophilic LAB was favored by anaerobic conditions, high quantities of CO2 , or both. At low Ar or N2 concentration (15%), the dominant bacteria were Pseudomonas spp., Enterobacteriaceae, and B. thermosphacta with dominance of the latter increasing during storage [74]. These authors noted the ability of Pseudomonas spp., considered aerobic bacteria, to grow with only residual amounts of O2 . A pretreatment of 3 hours with 100% CO2 prior to packaging under 70% CO2 –15% O2 –15% N2 improved the microbiological quality of the meat of raw chicken drumsticks and prolonged shelf life [31]. The Pseudomonas counts, as well as the total aerobic counts, were significantly lower after 7 and 12 days of storage when a CO2 pretreatment was applied. Such treatment had no additional effect on coliforms, which were undetectable after seven days of storage under MAP, whether or not a CO2 pretreatment was applied. Such an effect on the shelf life resulted from a better availability of CO2 in the headspace during storage because of the dissolution of CO2 in meat after the pretreatment. Although varying gas composition of MAP certainly acts on shelf life and spoilage of poultry cuts, no clear description on the effect of gas on the growth of bacterial at the level species has been reported. 4.4. Variability of Bacterial Contaminants in Marinated Chicken and with Various Additives The definition of marinade varies according to the country [46,75]. Marinades may be composed of a mixture of oil or salt and phosphates (in France and Spain, for instance) or a sauce with oil, organic acids, or spices, essential oil and thickener (Finland, China, and Italy). In all cases, marinades are associated with storage under different MAPs. The combination of oregano essential oil addition at 0.1% or 1% with MAP on the microbiological quality of chicken cuts has been studied [35]. The addition of 0.1% oregano essential oil increased the shelf life by 3–4 days, while the increase provided by the gas mixture (70% CO2 −30% N2 ) was only 2–3 days. The combination of a marinade with oregano essential oil and storage under MAP showed that the two treatments could be added as the shelf life reached more than 20 days with a decrease in the total viable count of 2–3 log CFU/g [35]. In Finland, the consumption of marinated poultry products packaged under MAP is common and the effect of the marinade on their microbial safety has been well documented. The Finnish marinade can be complex as it is composed of acetic acid, honey, glucose, maltodextrin, NaCl, phosphate, rape

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seed oil, spices (sweet pepper, curry, black pepper, garlic and turmeric), thickener (guar gum and xanthan gum), and yeast extract [7]. Such marinades may influence the LAB population by favoring the growth of specific species, particularly because of the source of carbohydrates they provide [46]. The MAP commonly used in Finland is composed of 65% N2 and 35% CO2 . The marinade favors a LAB psychrotrophic population, not detected in the unmarinated products [46]; especially L. gasicomitatum, also present in spoiled meat and seafood products [13] and in some vegetables associated with marinated fish products [76]. This bacterial species, unable to survive in the digestive tract of the animal, certainly originates from the environment and is adapted to the cold because it can persist throughout the transformation process [46]. As the combination of MAP and marinade favors the emergence of this group of bacteria, it is necessary to understand their mechanism of adaptation to monitor them in such products. It should be noted that the marinade had no effect on Campylobacter [46]. In a study combining the identification of isolates, as well as 16S rDNA gene pyrosequencing and metagenomics an overview of the effect of marinades on broiler fillet strip microbiology was reported [6]. Samples stored at 6 ◦ C under MAP (65% N2 −35% CO2 ) with and without marinade were compared. The combination of cultural and molecular methods confirmed that among LAB, marinade favored Leuconostoc and particularly L. gasicomitatum, and decreased B. thermosphacta, Clostridium spp., and Enterobacteriaceae. Among LAB belonging to the genus Carnobacterium, C. divergens was present in higher amounts than C. maltaromaticum, although both species seemed sensitive to marinade, certainly because of the presence of acetic acid. 4.5. Variability of Bacterial Contaminants Regarding Sanitizing Treatments The effect of several sanitizing treatments tested on in laboratory media or on meat cuts has also been assessed. These treatments are summarized in Table 4. Table 4. Examples of sanitizing treatments tested and experimental designs. Experimental Design

TSP 1

ASC

CA

PA

Laboratory conditions [77] 2

X3

X

X

X

[78] [79] Challenge-tests

X X

X X

X X

X

X

[80] [81] [82] [83] Natural contamination [37] [84] [85] [86] [36]

X X X X

X X X

X X X

X X X

X X

X

X

X

X

X X X

X X

X X

X

CD

LA

AA

X X

KP

KO

X

X

G

X X

1

TSP: trisodium phosphate, ASC: acidified sodium chlorite, CA: citric acid, PA: peroxy acids, CD: chlorine dioxide, LA: lactic acid, AA: acetic acid, KP: K3 PO4 , KO: potassium oleate, G: glutamal; 2 Examples were collected from references [36,37,77–86]; 3 Compound analyzed in the corresponding article.

In laboratory conditions (in vitro), the effect of three treatments on the lag phase and on the maximum growth rate was measured on several pathogenic (S. enterica serotype Enteritidis, L. monocytogenes) and spoilage (P. fluorescens and B. thermosphacta) bacteria [78]. Acidified sodium chlorite was the most effective at decreasing the growth of all tested bacteria, whereas trisodium phosphate and citric acid were more effective against Gram-negative and Gram-positive bacteria, respectively. However, the effectiveness varied with the concentrations used. For example, at low concentrations trisodium phosphate increased the growth rate of S. enterica and L. monocytogenes. As well as the consequence of the strong effect of citric acid toward B. thermosphacta, the possible

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increased growth of pathogens was questioned. Thus, the authors questioned the potential danger to consumers of some treatments, by increasing the proportion of pathogenic bacteria with regard to the spoilage ones. The same conclusion about the dangerous effects of treatments favoring pathogenic bacteria as an indirect consequence of inhibiting spoilage bacteria was shared by other authors [80]. In addition, the acid stress response of L. monocytogenes after exposure to acidic poultry meat decontaminants may even enhance its survival of a subsequent exposure to stronger acidity, such as that encountered during gastric transit [79]. This adaptation to acidic conditions involves membrane fluidity in L. monocytogenes and S. enterica and suggests that other decontaminants should be preferred rather than sub-inhibitory concentrations of citric acid or peroxy acids [77]. In summary, these studies showed that trisodium phosphate and citric acid were effective against Gram-positive pathogenic bacteria and peroxy acids and acidified sodium chlorite against Gram-negative bacteria. However, the observation of significant reductions in the microbial level immediately after treatment resulted from trials that were not performed in real meat conditions. Naturally contaminated meat matrices have also been used [36,37,84–86]. In these conditions, all decontaminants tested (trisodium phosphate, lactic and citric acids, peroxy acids, acidified sodium chlorite) reduced the total viable counts, Enterobacteriaceae, Pseudomonas, and LAB counts. The most effective concentrations reported were 14% for trisodium phosphate and 5% for citric acid. Trisodium phosphate, citric acid, acidified sodium chlorite, and peroxy acids were considered interesting treatments for extending the shelf life and improving the safety of products [37]. The effectiveness of chemical decontaminants and physical treatments (such as steam, hot water, and electricity) during or after the slaughtering process has been reviewed [87]. These authors emphasized that besides the relative effectiveness of treatments toward a variety of bacterial species, these must be considered as part of an integral food safety system. In that sense, some authors also completed the analysis of treatments of carcasses against pathogens with a sensory analysis performed by trained panelists on the cooked carcasses [85]. Since then, several other authors have also included the analysis of the sensory impact of decontamination treatments [88]. The impact of other physical decontamination processes on the microbiology of poultry meat has also been investigated. High hydrostatic pressure associated with the addition of nisin or glucono delta-lactone was effective at decreasing the counts of psychrotrophic bacteria and, to a lesser extent, mesophilic bacteria [89]. Gamma irradiation associated with storage under different MAPs was also effective at reducing LAB, B. thermosphacta, Pseudomonas, and Enterobacteriaceae [90]. Nevertheless, although such physical treatments have proven their ability to reduce the microbial load, they may have indirect effects on the sensory attributes of meat (color, texture). In addition, the perception by consumers of such practices can be controversial and their use is regulated differently depending on the country [91,92]. 5. Conclusions The poultry meat sector tends to provide ready to eat products, which are safe for the consumer and have a long shelf life. The biological hazards associated with poultry meat production and consumption have been well identified, ranking Campylobacter spp. and Salmonella spp. as a high risk [93]. Such ranking took the severity of the illnesses caused by these pathogens, their impact on human health, the number of cases, and the occurrence of the risk in the poultry meat production chain into account. Consequently, the impact of various treatments (temperature, chemical treatment, marinade, or preservation processes) in reducing pathogens has been investigated. Many studies have also been conducted to test such treatments for extending the shelf life and avoiding spoilage. The large number of publications dedicated to poultry meat microbiology and the variety of the results highlight the wide diversity of the microbiological status of poultry meat products. The bacterial loads can vary by several log CFU/g for similar cuts, stored under similar conditions. To date, the microbial ecology of poultry meat products has been considered mainly through cultural methods, which can introduce a bias because of the relative selectivity of the media used. In particular, poorly

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selective media targeting large families of bacteria such as LAB or Enterobacteriaceae have been used, leading to a poor characterization of the bacterial species present. The studies aiming at assessing the spoilage and/or shelf life of the products have used various criteria that make it difficult to describe clearly which bacteria can spoil poultry meat under which conditions, except for marinated poultry. In fact, marinades providing sugar and acetic acid lead to a pressure selection on the bacterial diversity, including bacteria responsible for spoilage, with the identification of the bacterial functions involved in spoilage appearance. Concerning pathogens, most of the efforts have focused on tracking them, while only a few describe their behavior in the meat matrix and consider the meat microbiota. In fact, two approaches can be distinguished: one focusing on only one or a few species, mostly pathogenic, with little attention paid to the microbiota because of the low contamination level of pathogens regarding that of total counts; and one focusing on a wider range of microbes, but assessing microbiota with techniques that induce a bias in the identification or that are generalist because of the media used. A third approach, already used for investigating complex environments, has recently appeared in food microbiology and tends to study the microbiota by non-cultural methods. The advantage of the latter is a better description of the bacterial species present in poultry meat, regardless of the detection of pathogens that are often present at a lower level. Finally, although the gastrointestinal tract of birds and slaughtering facilities have been identified as the main reservoirs for the origin of poultry meat contaminants, there is little knowledge about the flux of microbiota along the production chain from the animals to the end products. Indeed, the few studies about the transmission from animal to meat have mainly focused on pathogens. The combination of high throughput sequencing approaches with highly selective cultural methods throughout the production chain will be necessary to assess the sources of meat contaminants, their identification, and their dynamics during processing and storage. Moreover, metatranscriptomics may also be useful for determining the metabolic functions expressed by bacterial contaminants during meat processing and storage. Combining this with metabolomics should unravel the complex behavior of the poultry meat contaminants along the chain of food production. This should help to better manage meat ecosystems and improve the microbial quality and safety of food. Acknowledgments: This work was financed by the “Région Pays de la Loire” (PhD grant to AR) and the project was supported by Valorial, an agri-food competitiveness cluster. We thank Catherine Magras and Hervé Prévost, Professors at Oniris (Nantes, France) and Benoît Remenant, Project manager at Anses (Angers, France), for helpful discussions and Carol Robins for English reviewing. Conflicts of Interest: The authors declare no conflict of interest.

References 1. 2. 3. 4.

5. 6.

7.

Meat Consumption (Indicator). Available online: https://data.oecd.org/agroutput/meat-consumption.htm (accessed on 24 July 2017). Tout Savoir sur la Volaille Française. Available online: https://www.volaille-francaise.fr/la-filiere-avicole/ chiffres-cles/ (accessed on 24 July 2017). Filières Agricoles et de la Pêche et Aquaculture. Available online: http://www.franceagrimer.fr/ (accessed on 24 July 2017). Rouger, A.; Remenant, B.; Prévost, H.; Zagorec, M. A method to isolate bacterial communities and characterize ecosystems from food products: Validation and utilization as a reproducible chicken meat model. Int. J. Food Microbiol. 2017, 247, 38–47. [CrossRef] [PubMed] Sauter, E.A. Microbiology of frozen poultry products. In The Microbiology of Poultry Meat Products; Cunningham, F.E., Cox, N.A., Eds.; Academic Press: New York, NY, USA, 1987; pp. 333–339. Nieminen, T.T.; Koskinen, K.; Laine, P.; Hultman, J.; Sade, E.; Paulin, L.; Paloranta, A.; Johansson, P.; Björkroth, J.; Auvinen, P. Comparison of microbial communities in marinated and unmarinated broiler meat by metagenomics. Int. J. Food Microbiol. 2012, 157, 142–149. [CrossRef] [PubMed] Nieminen, T.T.; Valitalo, H.; Sade, E.; Paloranta, A.; Koskinen, K.; Björkroth, J. The effect of marination on lactic acid bacteria communities in raw broiler fillet strips. Front. Microbiol. 2012, 3, 376. [CrossRef] [PubMed]

Microorganisms 2017, 5, 50

8. 9. 10.

11.

12.

13.

14. 15.

16. 17. 18. 19. 20. 21. 22. 23.

24. 25.

26.

27. 28.

29.

12 of 16

Authority, E.F.S. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2015. EFSA. J. 2016, 14, 4634. Chai, S.J.; Cole, D.; Nisler, A.; Mahon, B.E. Poultry: The most common food in outbreaks with known pathogens, United States, 1998–2012. Epidemiol. Infect. 2017, 145, 316–325. [CrossRef] [PubMed] Praveen, P.K.; Debnath, C.; Shekhar, S.; Dalai, N.; Ganguly, S. Incidence of Aeromonas spp. infection in fish and chicken meat and its related public health hazards: A review. Vet. World. 2016, 9, 6–11. [CrossRef] [PubMed] Line, J.E.; Oakley, B.B.; Stern, N.J. Comparison of cumulative drip sampling with whole carcass rinses for estimation of Campylobacter species and quality indicator organisms associated with processed broiler chickens. Poult. Sci. 2013, 92, 218–224. [CrossRef] [PubMed] Mormile, M.R.; Oakley, B.B.; Morales, C.A.; Line, J.; Berrang, M.E.; Meinersmann, R.J.; Tillman, G.E.; Wise, M.G.; Siragusa, G.R.; Hiett, K.L.; Seal, B.S. The poultry-associated microbiome: Network analysis and farm-to-fork characterizations. PLoS ONE 2013, 8, e57190. Chaillou, S.; Chaulot-Talmon, A.; Caekebeke, H.; Cardinal, M.; Christieans, S.; Denis, C.; Desmonts, M.H.; Dousset, X.; Feurer, C.; Hamon, E.; et al. Origin and ecological selection of core and food-specific bacterial communities associated with meat and seafood spoilage. ISME J. 2015, 9, 1105–1118. [CrossRef] [PubMed] SLAUGHTERHOUSES. Available online: http://www.fao.org/WAIRDOCS/LEAD/X6114E/x6114e04. htm#b2--2.2.%20Poultry%20slaughtering%20process (accessed on 27 January 2017). Vihavainen, E.; Lundstrom, H.S.; Susiluoto, T.; Koort, J.; Paulin, L.; Auvinen, P.; Bjorkroth, J. Role of broiler carcasses and processing plant air in contamination of modified-atmosphere-packaged broiler products with psychrotrophic lactic acid bacteria. Appl. Environ. Microbiol. 2007, 73, 1136–1145. [CrossRef] [PubMed] Luber, P. Cross-contamination versus undercooking of poultry meat or eggs—Which risks need to be managed first? Int. J. Food Microbiol. 2009, 134, 21–28. [CrossRef] [PubMed] Warsow, C.R.; Orta-Ramirez, A.; Marks, B.P.; Ryser, E.T.; Booren, A.M. Single directional migration of Salmonella into marinated whole muscle turkey breast. J. Food Prot. 2008, 71, 153–156. [CrossRef] [PubMed] Arnold, J.W. Bacterial contamination on rubber picker fingers before, during, and after processing. Poult. Sci. 2007, 86, 2671–2675. [CrossRef] [PubMed] Arnold, J.W.; Yates. Interventions for control of Salmonella: Clearance of microbial growth from rubber picker fingers. Poult. Sci. 2009, 88, 1292–1298. [CrossRef] [PubMed] Veluz, G.A.; Pitchiah, S.; Alvarado, C.Z. Attachment of Salmonella serovars and Listeria monocytogenes to stainless steel and plastic conveyor belts. Poult. Sci. 2012, 91, 2004–2010. Álvarez-Astorga, M.; Capita, R.; Alonso-Calleja, C.; Moreno, B.; del Camino García-Fernández, M. Microbiological quality of retail chicken by-products in Spain. Meat Sci. 2002, 62, 45–50. [CrossRef] Göksoy, E.; Kirkan, S.; Kok, F. Microbiological quality of broiler carcasses during processing in two slaughterhouses in Turkey. Poult. Sci. 2004, 83, 1427–1432. [CrossRef] [PubMed] Russell, S.M. The Effect of an acidic, copper sulfate-based commercial sanitizer on indicator, pathogenic, and spoilage bacteria associated with broiler chicken carcasses when applied at various intervention points during poultry processing. Poult. Sci. 2008, 87, 1435–1440. [CrossRef] [PubMed] Demirok, E.; Veluz, G.; Stuyvenberg, W.V.; Castaneda, M.P.; Byrd, A.; Alvarado, C.Z. Quality and safety of broiler meat in various chilling systems. Poult. Sci. 2013, 92, 1117–1126. [CrossRef] [PubMed] Hue, O.; Allain, V.; Laisney, M.J.; Le Bouquin, S.; Lalande, F.; Petetin, I.; Rouxel, S.; Quesne, S.; Gloaguen, P.Y.; Picherot, M.; et al. Campylobacter contamination of broiler caeca and carcasses at the slaughterhouse and correlation with Salmonella contamination. Food Microbiol. 2011, 28, 862–868. [PubMed] Pacholewicz, E.; Swart, A.; Wagenaar, J.A.; Lipman, L.J.; Havelaar, A.H. Explanatory variables associated with Campylobacter and Escherichia coli concentrations on broiler chicken carcasses during processing in two slaughterhouses. J. Food Prot. 2016, 79, 2038–2047. [CrossRef] [PubMed] Waite, D.W.; Taylor, M.W. Characterizing the avian gutmicrobiota: membership, driving influences, and potential function. Front. Microbiol. 2014, 5, 223. [CrossRef] [PubMed] Mohd Shaufi, M.A.; Sieo, C.C.; Chong, C.W.; Gan, H.M.; Ho, Y.W.; Sunway, B. Deciphering chicken gut microbial dynamics based on high-throughput 16S rRNA metagenomics analyses. Gut Pathog. 2015, 7, 4. [CrossRef] [PubMed] Ranjitkar, S.; Lawley, B.; Tannock, G.; Engberg, R.M. Bacterial succession in the broiler gastrointestinal tract. Appl. Environ. Microbiol. 2016, 82, 2399–2410. [CrossRef] [PubMed]

Microorganisms 2017, 5, 50

30. 31.

32.

33. 34.

35.

36.

37.

38. 39.

40. 41.

42.

43.

44. 45.

46. 47.

48. 49.

13 of 16

Hinton, A., Jr.; Cason, J.A.; Ingram, K.D. Tracking spoilage bacteria in commercial poultry processing and refrigerated storage of poultry carcasses. Int. J. Food Microbiol. 2004, 91, 155–165. [CrossRef] Al-Nehlawi, A.; Saldo, J.; Vega, L.F.; Guri, S. Effect of high carbon dioxide atmosphere packaging and soluble gas stabilization pre-treatment on the shelf-life and quality of chicken drumsticks. Meat Sci. 2013, 94, 1–8. [CrossRef] [PubMed] Balamatsia, C.C.; Patsias, A.; Kontominas, M.G.; Savvaidis, I.N. Possible role of volatile amines as quality-indicating metabolites in modified atmosphere-packaged chicken fillets: Correlation with microbiological and sensory attributes. Food Chem. 2007, 104, 1622–1628. [CrossRef] Chaiba, A.; Rhazi, F.F.; Chahlaoui, A.; Soulaymani, R.B.; Zerhouni, M. Microbiological quality of poultry meat on the Meknès market (Morocco). Internet J. Food Safety 2007, 9, 67–71. Capita, R.; Alonso-Calleja, C.; García-Arias, M.T.; Moreno, B.; García-Fernández, M.D.C. Methods to detect the occurrence of various indicator bacteria on the surface of retail poultry in Spain. J. Food Sci. 2002, 67, 765–771. [CrossRef] Chouliara, E.; Karatapanis, A.; Savvaidis, I.N.; Kontominas, M.G. Combined effect of oregano essential oil and modified atmosphere packaging on shelf-life extension of fresh chicken breast meat, stored at 4 ◦ C. Food Microbiol. 2007, 24, 607–617. [CrossRef] [PubMed] Capita, R.; Álvarez-Fernández, E.; Fernández-Buelta, E.; Manteca, J.; Alonso-Calleja, C. Decontamination treatments can increase the prevalence of resistance to antibiotics of Escherichia coli naturally present on poultry. Food Microbiol. 2013, 34, 112–117. [CrossRef] [PubMed] Del Río, E.; Panizo-Morán, M.; Prieto, M.; Alonso-Calleja, C.; Capita, R. Effect of various chemical decontamination treatments on natural microflora and sensory characteristics of poultry. Int. J. Food Microbiol. 2007, 115, 268–280. [CrossRef] [PubMed] Arnaut-Rollier, I.; De Zutter, L.; Van Hoof, J. Identities of the Pseudomonas spp. in flora from chilled chicken. Int. J. Food Microbiol. 1999, 48, 87–96. [CrossRef] Arnaut-Rollier, I.; Vauterin, L.; De Vos, P.; Massart, D.L.; Devriese, L.A.; De Zutter, L.; Van Hoof, J.A. numerical taxonomic study of the Pseudomonas flora isolated from poultry meat. J. Appl. Microbiol. 1999, 87, 15–28. [CrossRef] [PubMed] Säde, E.; Murros, A.; Björkroth, J. Predominant enterobacteria on modified-atmosphere packaged meat and poultry. Food Microbiol. 2013, 34, 252–258. [CrossRef] [PubMed] Rahkila, R.; Johansson, P.; Säde, E.; Björkroth, J. Identification of enterococci from broiler products and a broiler processing plant and description of Enterococcus viikkiensis sp. nov. Appl. Environ. Microbiol. 2011, 77, 1196–1203. [CrossRef] [PubMed] Harada, T.; Dang, V.C.; Nguyen, D.P.; Nguyen, T.A.; Sakamoto, M.; Ohkuma, M.; Motooka, D.; Nakamura, S.; Uchida, K.; Jinnai, M.; et al. Enterococcus saigonensis sp. nov., isolated from retail chicken meat and liver. Int. J. Syst. Evol. Microbiol. 2016, 66, 3779–3785. [PubMed] Koort, J.; Murros, A.; Coneye, T.; Eerola, S.; Vandamme, P.; Sukura, A.; Björkroth, J. Lactobacillus oligofermentans sp. nov., associated with spoilage of modified-atmosphere-packaged poultry products. Appl. Environ. Microbiol. 2005, 71, 4400–4406. [CrossRef] [PubMed] Höll, L.; Behr, J.; Vogel, R.F. Identification and growth dynamics of meat spoilage microorganisms in modified atmosphere packaged poultry meat by MALDI-TOF MS. Food Microbiol. 2016, 60, 84–91. [CrossRef] [PubMed] Zhang, Q.Q.; Han, Y.Q.; Cao, J.X.; Xu, X.L.; Zhou, G.H.; Zhang, W.Y. The spoilage of air-packaged broiler meat during storage at normal and fluctuating storage temperatures. Poult. Sci. 2012, 91, 208–214. [CrossRef] [PubMed] Doulgeraki, A.I.; Ercolini, D.; Villani, F.; Nychas, G.-J.E. Spoilage microbiota associated to the storage of raw meat in different conditions. Int. J. Food Microbiol. 2012, 157, 130–141. [CrossRef] [PubMed] Wang, G.Y.; Wang, H.H.; Han, Y.W.; Xing, T.; Ye, K.P.; Xu, X.L.; Zhou, G.H. Evaluation of the spoilage potential of bacteria isolated from chilled chicken in vitro and in situ. Food Microbiol. 2017, 63, 139–146. [CrossRef] [PubMed] Björkroth, J. Microbiological ecology of marinated meat products. Meat Sci. 2005, 70, 477–480. [CrossRef] [PubMed] Andreevskaya, M.; Johansson, P.; Laine, P.; Smolander, O.-P.; Sonck, M.; Rahkila, R.; Jääskeläinen, E.; Paulin, L.; Auvinen, P.; Björkroth, J. Genome sequence and transcriptome analysis of meat-spoilage-associated lactic acid bacterium Lactococcus piscium MKFS47. Appl. Environ. Microbiol. 2015, 81, 3800–3811. [CrossRef] [PubMed]

Microorganisms 2017, 5, 50

50.

51.

52. 53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64. 65.

66.

67.

14 of 16

Jääskeläinen, E.; Johansson, P.; Kostiainen, O.; Nieminen, T.; Schmidt, G.; Somervuo, P.; Mohsina, M.; Vanninen, P.; Auvinen, P.; Björkroth, J. Significance of heme-based respiration in meat spoilage caused by Leuconostoc gasicomitatum. Appl. Environ. Microbiol. 2013, 79, 1078–1085. [CrossRef] [PubMed] Jääskeläinen, E.; Vesterinen, S.; Parshintsev, J.; Johansson, P.; Riekkola, M.-L.; Björkroth, J. Production of buttery-odor compounds and transcriptome response in Leuconostoc gelidum subsp. gasicomitatum LMG18811T during growth on various carbon sources. Appl. Environ. Microbiol. 2015, 81, 1902–1908. [PubMed] Grant, A.; Hashem, F.; Parveen, S. Salmonella and Campylobacter: Antimicrobial resistance and bacteriophage control in poultry. Food Microbiol. 2016, 53, 104–109. [CrossRef] [PubMed] Economou, V.; Zisides, N.; Gousia, P.; Petsios, S.; Sakkas, H.; Soultos, N.; Papadopoulou, C. Prevalence and antimicrobial profile of Campylobacter isolates from free-range and conventional farming chicken meat during a 6-year survey. Food Control 2015, 56, 161–168. [CrossRef] Fontanot, M.; Iacumin, L.; Cecchini, F.; Comi, G.; Manzano, M. Rapid detection and differentiation of important Campylobacter spp. in poultry samples by dot blot and PCR. Food Microbiol. 2014, 43, 28–34. [CrossRef] [PubMed] Oakley, B.B.; Morales, C.A.; Line, J.E.; Seal, B.S.; Hiett, K.L. Application of high-throughput sequencing to measure the performance of commonly used selective cultivation methods for the foodborne pathogen Campylobacter. FEMS Microbiol. Ecol. 2012, 79, 327–336. [CrossRef] [PubMed] Wassenaar, T.M.; Newell, D.G. The genus Campylobacter. In the Prokaryotes. Vol. 7: Proteobacteria: Delta and Epsilon Subclasses. Deeply Rooting Bacteria, 3rd ed.; Martin Dworkin, S.F., Ed.; Springer: New York, NY, USA, 2006; pp. 119–138. Jackson, B.R.; Griffin, P.M.; Cole, D.; Walsh, K.A.; Chai, S.J. Outbreak-associated Salmonella enterica serotypes and food commodities, United States, 1998–2008. Emerg. Infect. Dis. 2013, 19, 1239–1244. [CrossRef] [PubMed] Capita, R.; Alonso-Calleja, C.; Moreno, B.; García-Fernández, M.A.C. Occurrence of Listeria species in retail poultry meat and comparison of a cultural/immunoassay for their detection. Int. J. Food Microbiol. 2001, 65, 75–82. [CrossRef] Gudbjörnsdóttir, B.; Suihko, M.L.; Gustavsson, P.; Thorkelsson, G.; Salo, S.; Sjöberg, A.M.; Niclasen, O.; Bredholt, S. The incidence of Listeria monocytogenes in meat, poultry and seafood plants in the Nordic countries. Food Microbiol. 2004, 21, 217–225. [CrossRef] Van Nierop, W.; Duse, A.G.; Marais, E.; Aithma, N.; Thothobolo, N.; Kassel, M.; Stewart, R.; Potgieter, A.; Fernandes, B.; Galpin, J.S.; Bloomfield, S.F. Contamination of chicken carcasses in Gauteng, South Africa, by Salmonella, Listeria monocytogenes and Campylobacter. Int. J. Food Microbiol. 2005, 99, 1–6. [CrossRef] [PubMed] Cohen, N.; Ennaji, H.; Bouchrif, B.; Hassar, M.; Karib, H. Comparative study of microbiological quality of raw poultry meat at various seasons and for different slaughtering processes in Casablanca (Morocco). J. Appl. Poult. Res. 2007, 16, 502–508. [CrossRef] Alonso-Hernando, A.; Prieto, M.; García-Fernández, C.; Alonso-Calleja, C.; Capita, R. Increase over time in the prevalence of multiple antibiotic resistance among isolates of Listeria monocytogenes from poultry in Spain. Food Control 2012, 23, 37–41. [CrossRef] Waters, A.E.; Contente-Cuomo, T.; Buchhagen, J.; Liu, C.M.; Watson, L.; Pearce, K.; Foster, J.T.; Bowers, J.; Driebe, E.M.; Engelthaler, D.M.; Keim, P.S.; Price, L.B. Multidrug-resistant Staphylococcus aureus in US meat and poultry. Clin. Infect. Dis. 2011, 52, 1227–1230. [CrossRef] [PubMed] Akbar, A.; Anal, A.K. Prevalence and antibiogram study of Salmonella and Staphylococcus aureus in poultry meat. Asian Pac. J. Trop. Biomed. 2013, 3, 163–168. [CrossRef] Krupa, P.; Bystron, J.; Bania, J.; Podkowik, M.; Empel, J.; Mroczkowska, A. Genotypes and oxacillin resistance of Staphylococcus aureus from chicken and chicken meat in Poland. Poult. Sci. 2014, 93, 3179–3186. [CrossRef] [PubMed] Juneja, V.K.; Baker, D.A.; Thippareddi, H.; Snyder, O.P., Jr.; Mohr, TB. Growth potential of Clostridium perfringens from spores in acidified beef, pork and poultry products during chilling. J. Food Prot. 2013, 76, 65–71. [CrossRef] [PubMed] Mohr, T.B.; Juneja, V.K.; Thippareddi, H.H.; Schaffner, D.W.; Bronstein, P.A.; Silverman, M.; Cook, L.V., Jr. Assessing the performance of Clostridium perfringens cooling models for cooked, uncured meat and poultry products. J. Food Prot. 2015, 78, 1512–1526. [CrossRef] [PubMed]

Microorganisms 2017, 5, 50

68.

69. 70.

71. 72.

73.

74.

75. 76.

77.

78.

79.

80.

81.

82.

83.

84. 85. 86.

15 of 16

Huang, L. Evaluating the performance of a new model for predicting the growth of Clostridium perfringens in cooked, uncured meat and poultry products under isothermal, heating, and dynamically cooling conditions. J. Food Sci. 2016, 81, 1754–1765. [CrossRef] [PubMed] Tuncer, B.; Sireli, U.T. Microbial growth on broiler carcasses stored at different temperatures after air- or water-chilling. Poult. Sci. 2008, 87, 793–799. [CrossRef] [PubMed] Smolander, M.; Alakomi, H.-L.; Ritvanen, T.; Vainionpää, J.; Ahvenainen, R. Monitoring of the quality of modified atmosphere packaged broiler chicken cuts stored in different temperature conditions. A. Time-temperature indicators as quality-indicating tools. Food Control 2014, 15, 217–229. [CrossRef] Rossaint, S.; Klausmann, S.; Kreyenschmidt, J. Effect of high-oxygen and oxygen-free modified atmosphere packaging on the spoilage process of poultry breast fillets. Poult. Sci. 2015, 94, 93–103. [CrossRef] [PubMed] Balamatsia, C.C.; Paleologos, E.K.; Kontominas, M.G.; Savvaidis, I.N. Correlation between microbial flora, sensory changes and biogenic amines formation in fresh chicken meat stored aerobically or under modified atmosphere packaging at 4 degrees C: Possible role of biogenic amines as spoilage indicators. Antonie Van Leeuwenhoek 2006, 89, 9–17. [CrossRef] [PubMed] Patsias, A.; Chouliara, I.; Badeka, A.; Savvaidis, I.N.; Kontominas, M.G. Shelf-life of a chilled precooked chicken product stored in air and under modified atmospheres: Microbiological, chemical, sensory attributes. Food Microbiol. 2006, 23, 423–429. [CrossRef] [PubMed] Herbert, U.; Rossaint, S.; Khanna, M.-A.; Kreyenschmidt, J. Comparison of argon-based and nitrogen-based modified atmosphere packaging on bacterial growth and product quality of chicken breast fillets. Poult. Sci. 2013, 92, 1348–1356. [CrossRef] [PubMed] Yusop, S.M.; O’Sullivan, M.G.; Kerry, J.F.; Kerry, J.P. Effect of marinating time and low pH on marinade performance and sensory acceptability of poultry meat. Meat Sci. 2010, 85, 657–663. [CrossRef] [PubMed] Lyhs, U.; Koort, J.M.K.; Lundstrom, H.-S.; Björkroth, J. Leuconostoc gelidum and Leuconostoc gasicomitatum strains dominated the LAB population associated with strong slime formation in an acetic-acid herring preserve. Int. J. Food Microbiol. 2003, 90, 2207–2218. Alonso-Hernando, A.; Alonso-Calleja, C.; Capita, R. Effects of exposure to poultry chemical decontaminants on the membrane fluidity of Listeria monocytogenes and Salmonella enterica strains. Int. J. Food Microbiol. 2010, 137, 130–136. [CrossRef] [PubMed] Del Río, E.; González de Caso, B.; Prieto, M.; Alonso-Calleja, C.; Capita, R. Effect of poultry decontaminants concentration on growth kinetics for pathogenic and spoilage bacteria. Food Microbiol. 2008, 25, 888–894. [CrossRef] [PubMed] Alonso-Hernando, A.; Alonso-Calleja, C.; Capita, R. Comparative analysis of acid resistance in Listeria monocytogenes and Salmonella enterica strains before and after exposure to poultry decontaminants. Role of the glutamate decarboxylase (GAD) system. Food Microbiol. 2009, 26, 905–909. [CrossRef] [PubMed] Alonso-Hernando, A.; Capita, R.; Alonso-Calleja, C. Behaviour of co-inoculated pathogenic and spoilage bacteria on poultry following several decontamination treatments. Int. J. Food Microbiol. 2012, 159, 152–159. [CrossRef] [PubMed] Alonso-Hernando, A.; Guevara-Franco, J.A.; Alonso-Calleja, C.; Capita, R. Effect of the temperature of the dipping solution on the antimicrobial effectiveness of various chemical decontaminants against pathogenic and spoilage bacteria on poultry. J. Food Prot. 2013, 76, 833–842. [CrossRef] [PubMed] Del Río, E.; Muriente, R.; Prieto, M.; Alonso-Calleja, C.; Capita, R. Effectiveness of trisodium phosphate, acidified sodium chlorite, citric acid, and peroxy acids against pathogenic bacteria on poultry during refrigerated storage. J. Food Prot. 2007, 70, 2063–2071. [CrossRef] [PubMed] Del Río, E.; Capita, R.; Prieto, M.; Alonso-Calleja, C. Comparison of pathogenic and spoilage bacterial levels on refrigerated poultry parts following treatment with trisodium phosphate. Food Microbiol. 2006, 23, 195–198. [CrossRef] [PubMed] Hinton, A.; Ingram, K.D. Bactericidal activity of tripotassium phosphate and potassium oleate on the native flora of poultry skin. Food Microbiol. 2003, 20, 405–410. [CrossRef] Okolocha, E.C.; Ellerbroek, L. The influence of acid and alkaline treatments on pathogens and the shelf life of poultry meat. Food Control 2005, 16, 217–225. [CrossRef] Bolton, D.J.; Meredith, H.; Walsh, D.; McDowell, D.A. The effect of chemical treatments in laboratory and broiler plant studies on the microbial status and shelf-life of poultry. Food Control 2014, 36, 230–237. [CrossRef]

Microorganisms 2017, 5, 50

87. 88.

89. 90.

91. 92.

93.

16 of 16

Loretz, M.; Stephan, R.; Zweifel, C. Antimicrobial activity of decontamination treatments for poultry carcasses: A literature survey. Food Control 2010, 21, 791–804. [CrossRef] Samant, S.S.; Crandall, P.G.; O’Bryan, C.; Lingbeck, J.M.; Martin, E.M.; Seo, H.S. Sensory impact of chemical and natural antimicrobials on poultry products: A review. Poult. Sci. 2015, 88, 1699–1710. [CrossRef] [PubMed] Yuste, J.; Mor-Mur, M.; Capellas, M.; Guamis, B.; Pla, R. Microbiological quality of mechanically recovered poultry meat treated with high hydrostatic pressure and nisin. Food Microbiol. 1998, 15, 407–414. [CrossRef] Chouliara, E.; Badeka, A.; Savvaidis, I.; Kontominas, M.G. Combined effect of irradiation and modified atmosphere packaging on shelf-life extension of chicken breast meat: Microbiological, chemical and sensory changes. Eur. Food Res. Technol. 2008, 226, 877–888. [CrossRef] Ahn, D.U.; Kim, I.S.; Lee, E.J. Irradiation and additive combinations on the pathogen reduction and quality of poultry meat. Poult. Sci. 2013, 92, 534–545. [CrossRef] [PubMed] Directive 1999/2/EC of the European Parliament and of the Council of 22 February 1999 on the Approximation of the Laws of the Member States Concerning Foods and Food Ingredients Treated with Ionising Radiation. Available online: http://eur-lex.europa.eu/legal-content/EN/TXT/?qid= 1503584069651&uri=CELEX:01999L0002-20081211 (accessed on 26 July 2017). European Food Safety Authority (EFSA). Scientific Opinion on the public health hazards to be covered by inspection of meat (poultry). EFSA Panels on Biological Hazards (BIOHAZ), EFSA Panel on Contaminants in the Food Chain (CONTAM) and EFSA Panel on Animal Health and Welfare (AHAW). EFSA. J. 2012, 10, 2741. © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).